Nucleotide analogs

ABSTRACT

The disclosure provides nucleotide analogs and methods of their use. Analogs of the invention comprise a reporter molecule (label) attached via the N4, N6, O4, or O6 position of the nitrogenous base portion of the analog. In a preferred embodiment, nucleotide analogs of the invention comprise a label attached to the nitrogenous base portion of the analog via a cleavable linker at the N4, O4, N6 or O6 position.

BACKGROUND

Nucleic acid sequencing-by-synthesis has the potential to revolutionizethe understanding of biological structure and function. Traditionalsequencing technologies rely on amplification of sample-based nucleicacids and/or the use of electrophoretic gels in order to obtain sequenceinformation. More recently, single molecule sequencing has been proposedas a way to obtain high-throughput sequence information that is notsubject to amplification bias. See, Braslavsky, Proc. Natl. Acad. Sci.USA 100: 3960-64 (2003).

Sequencing-by-synthesis involves the template-dependent addition ofnucleotides to a support-bound template/primer duplex. The addednucleotides are labeled in a manner such that their incorporation intothe primer can be detected. A challenge that has arisen in singlemolecule sequencing involves the ability to sequence through homopolymerregions (i.e., portions of the template that contain consecutiveidentical nucleotides). Often the number of bases present in ahomopolymer region is important from the point of view of geneticfunction. As most polymerases used in sequencing-by-synthesis reactionsare highly-processive, they tend to add bases continuously as thepolymerase traverses a homopolymer region. Most detectable labels usedin sequencing reactions do not discriminate between more than twoconsecutive incorporations. Thus, a homopolymer region will be reportedas a single, or sometimes a double, incorporation without the resolutionnecessary to determine the exact number of bases present in thehomopolymer.

One solution to the problem of determining the number of bases presentin a homopolymer is proposed in co-owned U.S. Pat. No. 7,169,560. Thatmethod involves controlling the kinetics of the incorporation reactionsuch that, on average, only a predetermined number of bases areincorporated in any given reaction cycle. The present invention providesan alternative solution to this problem.

DESCRIPTION OF THE DRAWING

FIG. 1 shows the five common nitrogenous bases and the N6, O6, N4, andO4 positions, respectively.

SUMMARY OF THE INVENTION

The invention provides nucleotide analogs and methods of their use.Analogs of the invention comprise a reporter molecule (label) attachedvia the N4, N6, O4, or O6 position of the nitrogenous base portion ofthe analog.

In a preferred embodiment, nucleotide analogs of the invention comprisea label attached to the nitrogenous base portion of the analog via acleavable linker at the N4, O4, N6 or O6 position. Analogs can comprisemodifications at the nitrogenous base, sugar or phosphate. For example,analogs of the invention may be mono-, di-, or tri-phosphates, or may besubstituted phosphates, such as difluoro, dichloro, and others.Similarly, the sugar portion of the analog may be deoxy analogs, dideoxyanalogs, and may comprise a blocking moiety or other substitutions knownto those of skill in the art. Additionally, substituted or altered basestructures are contemplated by the invention.

In general, analogs of the invention may be placed into two broadcategories. Those based upon adenine or guanine (or their analogs) havethe general formula:

Wherein Y and X are independently selected from a cleavable bond,(CH₂)_(n) and (CH₂—O—CH₂)_(n), where, in each case n is from about 1 toabout 20 atoms.

Certain analogs based upon cytosine or thymine, uracil (or theiranalogs) have the general formula:

In each case, the nucleotide (which may be in the mono-, di-, ortriphosphate form) is linked at the N4, N6, O4, or O6 position via acleavable linker to a reporter or label. There are numerous linkerstructures that function in accordance with the invention to connect thenucleotide to the label. For example, the linker can be a straight-chainor branched chain alkyl, an ether, an ester, an aryl, or any combinationof the foregoing. In particularly-preferred embodiments, the linker isselected from the following structures, Structure 1 and Structure 2(where X represents the N6 or O6 position on the nucleotide; Yrepresents O, (CH₂)_(n), where n=1-10, or N; and Z represents O, N, analkyl, or an aryl group):

As the invention relates to improved structures having the linkerattached at the N4, N6, O4, or O6 position, the precise structure of thelinker and the label used are not of primary importance. Thus, anyconvenient linker and label can be applied at the convenience the user.

Preferred analogs of the invention are shown below:

Structure 3 is modified to include a label, in this case cyanine 5, asshown in Structure 4 above.

In another embodiment, an analog of the invention can take the formshown below in Structure 5:

In another exemplary embodiment, an O6-linked analog of the invention ispresented below in Structure 6:

Other exemplary structures according to the invention are shown below:

Thus, in one aspect, the invention provides a family of nucleotideanalogs, each having a label attached at the N4, N6 or O4, O6 position,depending on the identity of the nitrogenous base portion as discussedabove. In a preferred embodiment, the label is attached via a linker,preferably a cleavable linker, that is attached at the N4, N6 O4, or O6position of the nitrogenous base portion of the nucleotide.

The linker can be any chemical entity that can serve to connect the baseand the label. However, in a preferred embodiment, the linker isselected from alkyl or aryl groups. The linker can be from about 5 toabout 100 atoms. Any cleavable linkage can be used to remove the linkerand/or label from the nitrogenous base. Preferred cleavable groupsinclude a disulfide bond, amide bond, thioamide, bond, ester bond,thioester bond, vicinal diol bond, or hemiacetal. Other cleavable bondsinclude enzymatically-cleavable bonds, such as peptide bonds (cleaved bypeptidases), phosphate bonds (cleaved by phosphatases), nucleic acidbonds (cleaved by endonucleases), and sugar bonds (cleaved byglycosidases).

Analogs of the invention are reversible inhibitors or blockers thatallow the incorporation of only one nucleotide per addition cycle in atemplate-dependent sequencing-by-synthesis reaction. The compositionsdescribed herein are useful in any sequencing reaction, but areespecially useful in single molecule sequencing-by-synthesis reactions.Single molecule reactions are those in which the duplex to whichnucleotides are added is individually optically resolvable.

The nitrogenous base portion of the nucleotide is selected from thestandard Watson-Crick bases and their analogs and variants or analogs.In a specific embodiment, the invention provides a nucleotide analogcomprising a nucleotide to be incorporated linked to a blockingnucleotide comprising a traditional Watson-Crick base (adenine,guanosine, cytosine, thymidine, or uridine), a sugar for example, aribose or deoxy ribose sugar, and at least one phosphate.

The invention also provides methods for sequencing nucleic acids. Incertain methods, a nucleic acid duplex, comprising a template and aprimer, is positioned on a surface such that the duplex is individuallyoptically resolvable. A sequencing-by-synthesis reaction is performedunder conditions to permit addition of the labeled nucleotide analog tothe primer while preventing another nucleotide or nucleotide analog frombeing added immediately downstream. After incorporation has beendetected, inhibition is removed to permit another nucleotide to be addedto the primer. Methods of the invention allow, among other things,detection and counting of consecutive nucleotides in a templatehomopolymer region.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides nucleotide analogs that useful insequencing-by-synthesis reactions. Analogs of the invention comprise alinker from the N6 or O6 position on the base portion of the nucleotideto a label, a blocker, or both. Thus, in one embodiment, an analog ofthe invention comprises a blocking group, which may be the label itself,attached via a linker or tether to the N6 or O6 position of the base,depending upon the identity of the base as described above. Blockinggroups attached to analogs of the invention allowsequencing-by-synthesis reactions to occur such that only one nucleotideaddition is made before subsequent additions are made to the template.This allows for sequencing through homopolymer regions (regions of thetemplate having repeats of the same nucleotide) one base at a timewhich, in turn, enables counting the number of nucleotides in ahomopolymer.

Analogs of the invention are based upon any of the standard Watson-Cricknucleotides or their variants, and may be in the mono-, di-, tri-, orbis phosphate configuration. The analogs can contain traditional riboseor deoxyribose sugar groups or non-traditional groups such as an acyNTPconstruct and others known to those skilled in the art. Analogs of theinvention comprise an N4, N6 or O4, O6-linked blocker or label to enabledetection of the analog upon incorporation in a sequencing-by-synthesisreaction.

As discussed above, the invention provides improved methods forsequencing a nucleic acid containing a homopolymer region. The methodcomprises exposing a nucleic acid template/primer duplex to (i) apolymerase which catalyzes nucleotide addition to the primer, and (ii) alabeled nucleotide triphosphate analog comprising a first nucleotide ora first nucleotide analog covalently bonded through a tether to aninhibitor under conditions that permit the polymerase to add the labelednucleotide triphosphate analog to the primer at a position complementaryto the first base in the template while preventing another nucleotide ornucleotide analog from being added to the primer at a positioncomplementary to the next downstream base. After the exposing step, thenucleotide triphosphate analog incorporated into the primer is detected.The inhibitor is removed to permit other nucleotides to be incorporatedinto the primer. It is contemplated that the label, for example, one ofthe optically detectable labels described herein, can be removed at thesame time as the inhibitor. Any of the tethered nucleotide analogsdescribed herein can be used in this type of sequencing protocol.

The following sections discuss general considerations for nucleic acidsequencing, for example, template considerations, polymerases useful insequencing-by-synthesis, choice of surfaces, reaction conditions, signaldetection and analysis.

Exemplary Synthesis

The following synthetic pathway was used to produce a compound of theinvention:

Nucleic Acid Templates

Nucleic acid templates include deoxyribonucleic acid (DNA) and/orribonucleic acid (RNA). Nucleic acid templates can be synthetic orderived from naturally occurring sources. In one embodiment, nucleicacid template molecules are isolated from a biological sample containinga variety of other components, such as proteins, lipids and non-templatenucleic acids. Nucleic acid template molecules can be obtained from anycellular material, obtained from an animal, plant, bacterium, fungus, orany other cellular organism. Biological samples for use in the presentinvention include viral particles or preparations. Nucleic acid templatemolecules can be obtained directly from an organism or from a biologicalsample obtained from an organism, e.g., from blood, urine, cerebrospinalfluid, seminal fluid, saliva, sputum, stool and tissue. Any tissue orbody fluid specimen may be used as a source for nucleic acid for use inthe invention. Nucleic acid template molecules can also be isolated fromcultured cells, such as a primary cell culture or a cell line. The cellsor tissues from which template nucleic acids are obtained can beinfected with a virus or other intracellular pathogen. A sample can alsobe total RNA extracted from a biological specimen, a cDNA library,viral, or genomic DNA.

Nucleic acid obtained from biological samples typically is fragmented toproduce suitable fragments for analysis. In one embodiment, nucleic acidfrom a biological sample is fragmented by sonication. Nucleic acidtemplate molecules can be obtained as described in U.S. PatentApplication Publication Number US2002/0190663 A1, published Oct. 9,2003. Generally, nucleic acid can be extracted from a biological sampleby a variety of techniques such as those described by Maniatis, et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., pp.280-281 (1982). Generally, individual nucleic acid template moleculescan be from about 5 bases to about 20 kb. Nucleic acid molecules may besingle-stranded, double-stranded, or double-stranded withsingle-stranded regions (for example, stem- and loop-structures).

A biological sample as described herein may be homogenized orfractionated in the presence of a detergent or surfactant. Theconcentration of the detergent in the buffer may be about 0.05% to about10.0%. The concentration of the detergent can be up to an amount wherethe detergent remains soluble in the solution. In a preferredembodiment, the concentration of the detergent is between 0.1% to about2%. The detergent, particularly a mild one that is nondenaturing, canact to solubilize the sample. Detergents may be ionic or nonionic.Examples of nonionic detergents include triton, such as the Triton® Xseries (Triton® X-100 t-Oct-C₆H₄—(OCH₂—CH₂)_(x)OH, x=9-10, Triton®X-100R, Triton® X-114 x=7-8), octyl glucoside, polyoxyethylene(9)dodecylether, digitonin, IGEPAL® CA630 octylphenyl polyethylene glycol,n-octyl-beta-D-glucopyranoside (betaOG), n-dodecyl-beta, Tween® 20polyethylene glycol sorbitan monolaurate, Tween® 80 polyethylene glycolsorbitan monooleate, polidocanol, n-dodecyl beta-D-maltoside (DDM),NP-40 nonylphenyl polyethylene glycol, C12E8 (octaethylene glycoln-dodecyl monoether), hexaethyleneglycol mono-n-tetradecyl ether(C14EO6), octyl-beta-thioglucopyranoside (octyl thioglucoside, OTG),Emulgen, and polyoxyethylene 10 lauryl ether (C12E10). Examples of ionicdetergents (anionic or cationic) include deoxycholate, sodium dodecylsulfate (SDS), N-lauroylsarcosine, and cetyltrimethylammoniumbromide(CTAB). A zwitterionic reagent may also be used in the purificationschemes of the present invention, such as Chaps, zwitterion 3-14, and3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulf-onate. It iscontemplated also that urea may be added with or without anotherdetergent or surfactant.

Lysis or homogenization solutions may further contain other agents, suchas reducing agents. Examples of such reducing agents includedithiothreitol (DTT), β-mercaptoethanol, DTE, GSH, cysteine, cysteamine,tricarboxyethyl phosphine (TCEP), or salts of sulfurous acid.

Nucleic Acid Polymerases

Nucleic acid polymerases generally useful in the invention include DNApolymerases, RNA polymerases, reverse transcriptases, and mutant oraltered forms of any of the foregoing. DNA polymerases and theirproperties are described in detail in, among other places, DNAReplication 2nd edition, Kornberg and Baker, W. H. Freeman, New York,N.Y. (1991). Known conventional DNA polymerases useful in the inventioninclude, but are not limited to, Pyrococcus furiosus (Pfu) DNApolymerase (Lundberg et al., 1991, Gene, 108: 1, Stratagene), Pyrococcuswoesei (Pwo) DNA polymerase (Hinnisdaels et al., 1996, Biotechniques,20:186-8, Boehringer Mannheim), Thermus thermophilus (Tth) DNApolymerase (Myers and Gelfand 1991, Biochemistry 30:7661), Bacillusstearothermophilus DNA polymerase (Stenesh and McGowan, 1977, BiochimBiophys Acta 475:32), Thermococcus litoralis (Tli) DNA polymerase (alsoreferred to as Vent™ DNA polymerase, Cariello et al., 1991,Polynucleotides Res, 19: 4193, New England Biolabs), 9°Nm™ DNApolymerase (New England Biolabs), Stoffel fragment, ThermoSequenase®(Amersham Pharmacia Biotech UK), Therminator™ (New England Biolabs),Thermotoga maritima (Tma) DNA polymerase (Diaz and Sabino, 1998 Braz J.Med. Res, 31:1239), Thermus aquaticus (Taq) DNA polymerase (Chien etal., 1976, J. Bacteoriol, 127: 1550), DNA polymerase, Pyrococcuskodakaraensis KOD DNA polymerase (Takagi et al., 1997, Appl. Environ.Microbiol. 63:4504), JDF-3 DNA polymerase (from thermococcus sp. JDF-3,Patent application WO 0132887), Pyrococcus GB-D (PGB-D) DNA polymerase(also referred as Deep Vent™ DNA polymerase, Juncosa-Ginesta et al.,1994, Biotechniques, 16:820, New England Biolabs), UlTma DNA polymerase(from thermophile Thermotoga maritima; Diaz and Sabino, 1998 Braz J.Med. Res, 31:1239; PE Applied Biosystems), Tgo DNA polymerase (fromthermococcus gorgonarius, Roche Molecular Biochemicals), E. coli DNApolymerase I (Lecomte and Doubleday, 1983, Polynucleotides Res.11:7505), T7 DNA polymerase (Nordstrom et al., 1981, J. Biol. Chem.256:3112), and archaeal DP1I/DP2 DNA polymerase II (Cann et al, 1998,Proc. Natl. Acad. Sci. USA 95:14250).

Both mesophilic polymerases and thermophilic polymerases arecontemplated. Thermophilic DNA polymerases include, but are not limitedto, ThermoSequenase®, 9°Nm™, Therminator™, Taq, Tne, Tma, Pfu, Tfl, Tth,Tli, Stoffel fragment, Vent™ and Deep Vent™ DNA polymerase, KOD DNApolymerase, Tgo, JDF-3, and mutants, variants and derivatives thereof. Ahighly-preferred form of any polymerase is a 3′ exonuclease-deficientmutant.

Reverse transcriptases useful in the invention include, but are notlimited to, reverse transcriptases from HIV, HTLV-1, HTLV-II, FeLV, FIV,SIV, AMV, MMTV, MoMuLV and other retroviruses (see Levin, Cell 88:5-8(1997); Verma, Biochim Biophys Acta. 473:1-38 (1977); Wu et al., CRCCrit. Rev Biochem. 3:289-347 (1975)).

Surfaces

In a preferred embodiment, nucleic acid template molecules are attachedto a substrate (also referred to herein as a surface) and subjected toanalysis by single molecule sequencing as described herein. Nucleic acidtemplate molecules are attached to the surface such that thetemplate/primer duplexes are individually optically resolvable.Substrates for use in the invention can be two- or three-dimensional andcan comprise a planar surface (e.g., a glass slide) or can be shaped. Asubstrate can include glass (e.g., controlled pore glass (CPG)), quartz,plastic (such as polystyrene (low cross-linked and high cross-linkedpolystyrene), polycarbonate, polypropylene and poly(methymethacrylate)),acrylic copolymer, polyamide, silicon, metal (e.g.,alkanethiolate-derivatized gold), cellulose, nylon, latex, dextran, gelmatrix (e.g., silica gel), polyacrolein, or composites.

Suitable three-dimensional substrates include, for example, spheres,microparticles, beads, membranes, slides, plates, micromachined chips,tubes (e.g., capillary tubes), microwells, microfluidic devices,channels, filters, or any other structure suitable for anchoring anucleic acid. Substrates can include planar arrays or matrices capableof having regions that include populations of template nucleic acids orprimers. Examples include nucleoside-derivatized CPG and polystyreneslides; derivatized magnetic slides; polystyrene grafted withpolyethylene glycol, and the like.

Substrates are preferably coated to allow optimum optical processing andnucleic acid attachment. Substrates for use in the invention can also betreated to reduce background. Exemplary coatings include epoxides, andderivatized epoxides (e.g., with a binding molecule, such as anoligonucleotide or streptavidin).

Various methods can be used to anchor or immobilize the nucleic acidmolecule to the surface of the substrate. The immobilization can beachieved through direct or indirect bonding to the surface. The bondingcan be by covalent linkage. See, Joos et al., Analytical Biochemistry247:96-101, 1997; Oroskar et al., Clin. Chem. 42:1547-1555, 1996; andKhandjian, Mol. Bio. Rep. 11:107-115, 1986. A preferred attachment isdirect amine bonding of a terminal nucleotide of the template or the 5′end of the primer to an epoxide integrated on the surface. The bondingalso can be through non-covalent linkage. For example,biotin-streptavidin (Taylor et al., J. Phys. D. Appl. Phys. 24:1443,1991) and digoxigenin with anti-digoxigenin (Smith et al., Science253:1122, 1992) are common tools for anchoring nucleic acids to surfacesand parallels. Alternatively, the attachment can be achieved byanchoring a hydrophobic chain into a lipid monolayer or bilayer. Othermethods for known in the art for attaching nucleic acid molecules tosubstrates also can be used.

Detection

Any detection method can be used that is suitable for the type of labelemployed. Thus, exemplary detection methods include radioactivedetection, optical absorbance detection, e.g., UV-visible absorbancedetection, optical emission detection, e.g., fluorescence orchemiluminescence. For example, extended primers can be detected on asubstrate by scanning all or portions of each substrate simultaneouslyor serially, depending on the scanning method used. For fluorescencelabeling, selected regions on a substrate may be serially scannedone-by-one or row-by-row using a fluorescence microscope apparatus, suchas described in Fodor (U.S. Pat. No. 5,445,934) and Mathies et al. (U.S.Pat. No. 5,091,652). Devices capable of sensing fluorescence from asingle molecule include scanning tunneling microscope (siM) and theatomic force microscope (AFM). Hybridization patterns may also bescanned using a CCD camera (e.g., Model TE/CCD512SF, PrincetonInstruments, Trenton, N.J.) with suitable optics (Ploem, in Fluorescentand Luminescent Probes for Biological Activity Mason, T. G. Ed.,Academic Press, Landon, pp. 1-11 (1993), such as described in Yershov etal., Proc. Natl. Acad. Sci. 93:4913 (1996), or may be imaged by TVmonitoring. For radioactive signals, a phosphorimager device can be used(Johnston et al., Electrophoresis, 13:566, 1990; Drmanac et al.,Electrophoresis, 13:566, 1992; 1993). Other commercial suppliers ofimaging instruments include General Scanning Inc., (Watertown, Mass. onthe World Wide Web at genscan.com), Genix Technologies (Waterloo,Ontario, Canada; on the World Wide Web at confocal.com), and AppliedPrecision Inc. Such detection methods are particularly useful to achievesimultaneous scanning of multiple attached template nucleic acids.

A number of approaches can be used to detect incorporation offluorescently-labeled nucleotides into a single nucleic acid molecule.Optical setups include near-field scanning microscopy, far-fieldconfocal microscopy, wide-field epi-illumination, light scattering, darkfield microscopy, photoconversion, single and/or multiphoton excitation,spectral wavelength discrimination, fluorophor identification,evanescent wave illumination, and total internal reflection fluorescence(TIRF) microscopy. In general, certain methods involve detection oflaser-activated fluorescence using a microscope equipped with a camera.Suitable photon detection systems include, but are not limited to,photodiodes and intensified CCD cameras. For example, an intensifiedcharge couple device (ICCD) camera can be used. The use of an ICCDcamera to image individual fluorescent dye molecules in a fluid near asurface provides numerous advantages. For example, with an ICCD opticalsetup, it is possible to acquire a sequence of images (movies) offluorophores.

Some embodiments of the present invention use TIRF microscopy forimaging. TIRF microscopy uses totally internally reflected excitationlight and is well known in the art. See, e.g., the World Wide Web atnikon-instruments.jp/eng/page/products/tirf.aspx. In certainembodiments, detection is carried out using evanescent wave illuminationand total internal reflection fluorescence microscopy. An evanescentlight field can be set up at the surface, for example, to imagefluorescently-labeled nucleic acid molecules. When a laser beam istotally reflected at the interface between a liquid and a solidsubstrate (e.g., a glass), the excitation light beam penetrates only ashort distance into the liquid. The optical field does not end abruptlyat the reflective interface, but its intensity falls off exponentiallywith distance. This surface electromagnetic field, called the“evanescent wave”, can selectively excite fluorescent molecules in theliquid near the interface. The thin evanescent optical field at theinterface provides low background and facilitates the detection ofsingle molecules with high signal-to-noise ratio at visible wavelengths.

The evanescent field also can image fluorescently-labeled nucleotidesupon their incorporation into the attached template/primer complex inthe presence of a polymerase. Total internal reflectance fluorescencemicroscopy is then used to visualize the attached template/primer duplexand/or the incorporated nucleotides with single molecule resolution.

Analysis

Alignment and/or compilation of sequence results obtained from the imagestacks produced as generally described above utilizes look-up tablesthat take into account possible sequences changes (due, e.g., to errors,mutations, etc.). Essentially, sequencing results obtained as describedherein are compared to a look-up type table that contains all possiblereference sequences plus 1 or 2 base errors.

1. A nucleotide analog, comprising: A nitrogenous base; A linkerattached at the N4, N6, O4, or O6 of said nitrogeneous base; and Adetectable label attached to said nitrogenous base via said linker,wherein said linker comprises a cleavable bond such that a nativenucleotide monophosphate is regenerated upon incorporation of saidanalog into a nucleic acid duplex.
 2. The nucleotide analog of claim 1,further comprising a mono-, di-, tri-, or bis-phosphate.
 3. Thenucleotide analog of claim 1, further comprising a ribose sugar or adeoxyribose sugar.
 4. The nucleotide analog of claim 1, wherein saidanalog has the structure:

Wherein P is a monophosphate, diphosphate, bisphosphate, triphosphate,or halogen-substituted phosphate, and wherein X and Y are independentlyselected from the group consisting of a cleavable bond, an alkyl, anesther, an ether, or a combination of foregoing.
 5. The nucleotideanalog of claim 4, wherein X is an alkyl group or an aryl group.
 6. Thenucleotide analog of claim 4, wherein said alkyl group comprises betweenabout 2 and about 10 atoms.
 7. The nucleotide analog of claim 1, whereinsaid label is selected from cyanine-5, Cy-5, Cy-3, Joe, Rox, and TAMRA.8. The nucleotide analog of claim 1, wherein said analog has thestructure:

Wherein P is a monophosphate, diphosphate, bisphosphate, triphosphate,or halogen-substituted phosphate, and wherein X and Y are independentlyselected from the group consisting of a cleavable bond, an alkyl, anesther, an ether, a halogen, or a combination of the foregoing.
 9. Thenucleotide analog of claim 8, wherein X is (CH₂)_(n), and wherein n isbetween about 2 and about
 10. 10. The nucleotide analog of claim 8,wherein Y is a carbonyl.
 11. The nucleotide analog of claim 8, whereinsaid label is an optically-detectable label.
 12. The nucleotide analogof claim 11, wherein said detectable label is selected from cyanine-5,Cy-5, Cy-3, Joe, Rox, and TAMRA.
 13. A method for sequencing a nucleicacid, the method comprising the steps of: anchoring a nucleic acidduplex to a surface, the duplex comprising a template portion and aprimer portion hybridized thereto: Exposing said duplex to an analog ofclaim 4 or claim 10 in the presence of a polymerase capable ofcatalyzing addition of said analog to said primer portion in atemplate-dependent manner; Removing unincorporated analog andpolymerase; Detecting incorporation of analog into said primer; andRepeating said exposing, removing, and detecting steps at least once.14. The method of claim 13, further comprising the step of cleavingdetectable label from said analog after said detecting step.